PROJECT SUMMARY/ ABSTRACT Thyroid hormone (TH) deficiency impacts vertebrate physiology in a myriad of ways. The pediatric consequences of congenital hypothyroidism (the most common congenital endocrinopathy worldwide) include defective growth and hearing, and an inability to achieve maximal potential intelligence. In adults, hypothyroidism leads to hyperlipidemia, altered thermogenesis, and weight gain promoting obesity. Throughout the entire vertebrate subphylum, THs are synthesized within the precursor protein thyroglobulin (Tg), encoded by the single TG gene, under the regulation of thyroid-stimulating hormone (TSH). Thyroxine (T4) derives primarily from a unique, evolutionarily conserved site at the N-terminus of Tg (on position Tyr-5), whereas the primary formation site for triiodothyronine (T3) derives from the opposite end of the Tg protein. Iodination of Tg is the sole source of T4 in the body, but only a partial contributor to circulating T3 (the remaining T3 is converted by deiodinating T4 in various organs). Treatment of hypothyroidism with T4 leaves many patients with persistent hypothyroid symptoms, whereas direct treatment exclusively with T3 results in dramatic up-down swings in blood levels of T3. I have been interested to know, what would be the consequences if the thyroid gland itself, regulated moment-to-moment by TSH, could selectively produce all of the body’s T3? Under physiological regulation by TSH, the carboxyl-terminal ChEL domain of Tg — by providing essentially exclusively T3 to the body — either will or will not be sufficient to sustain all critical developmental and metabolic functions supported by TH. To test this I propose to use CRISPR/Cas9-mutagenesis to develop the first two genetically-edited mouse models with homozygous TG knock-in mutants encoding either: a) a Tg-Y5F substitution (eliminating the primary T4-forming site of Tg) or b) encoding only the secretory ChEL domain that preserves the primary T3-forming site of Tg — in both cases leaving the primary T3-forming site of Tg intact in a thyroid gland physiologically regulated by TSH. With these novel animals in hand, I will study development, growth, behavior, hearing, body composition and weight, and thermoregulation. I will seek rigorous validation of in vivo hormonogesis studies with parallel cell culture experiments to express the same Tg variants, and characterize quantitatively their deficiency of T4 production but competence for T3 production, using Tg iodination in vitro, followed by nano-liquid chromatography tandem-mass spectrometry. The plan outlined in this 5-year K01 Award will permit me to develop additional expertise in whole animal thyroid pathophysiology as well as cutting edge biochemical skills needed for my future independent scientific career. Furthermore, this proposal is of significant clinical relevance to understand if there is any special role of T4 from neonates to adults, while providing deep insight into the evolutionary origin...